CN109781702A - A kind of magnetic microsphere and its preparation method and the detection method of microorganism - Google Patents
A kind of magnetic microsphere and its preparation method and the detection method of microorganism Download PDFInfo
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Abstract
本发明涉及微生物检测领域，具体而言，涉及一种磁性微球及其制备方法以及微生物的检测方法。一种磁性微球，包括微球本体，所述微球本体携带负电荷；吸附在所述微球本体外表面的携带正电荷的PEI。本发明提供的磁性微球能有效吸附样品中的微生物，特异性强，吸附较为紧密，降低其他杂质的干扰，通过检测拉曼信号使得不同微生物得以区分，该检测方法大大缩短检测时间，方法便捷可靠，准确率均为96％以上，为临床血液感染的诊断和治疗提供一种新颖、快速的检测方式。
The invention relates to the field of microorganism detection, in particular, to a magnetic microsphere and a preparation method thereof and a microorganism detection method. A magnetic microsphere, comprising a microsphere body, the microsphere body carrying a negative charge; and PEI carrying a positive charge adsorbed on the outer surface of the microsphere body. The magnetic microspheres provided by the invention can effectively adsorb the microorganisms in the sample, have strong specificity, relatively tight adsorption, reduce the interference of other impurities, distinguish different microorganisms by detecting Raman signals, the detection method greatly shortens the detection time, and the method is convenient It is reliable and has an accuracy rate of more than 96%, and provides a novel and rapid detection method for the diagnosis and treatment of clinical blood infection.
Description
Technical field
The present invention relates to microorganism detection field, in particular to a kind of magnetic microsphere and preparation method thereof and micro-
The detection method of biology.
Background technique
In recent years, with greatly developing for invasive diagnostic and treatment technology and answering extensively for broad-spectrum antibiotic and hormone
With the disease incidence of bloodstream infection increases year by year.The death rate of bloodstream infection is high, and disease incidence is high, and the hospital stays is long, medical expense
Height, harm are serious.Therefore, the control of bloodstream infection receives more and more attention.With the development of various operating technologies and anti-
The application of bacterium drug causes the pathogen of bloodstream infection constantly to change, and the drug resistance of pathogen gradually increases.Methicillin-resistant gold
Staphylococcus aureus (MRSA), Carbapenem-resistant pseudomonas aeruginosa (CRPA), Carbapenem-resistant Acinetobacter bauamnnii (CRAB)
It is continuously emerged with other antibody-resistant bacterium.Staphylococcus aureus, especially MRSA are that the most important of bloodstream infection is caused to cause a disease
One of bacterium.In severe cases, it may cause the fatal diseases such as pneumonia, infectious endocarditis, septicemia and osteomyelitis.
With the increase of clinical Antibiogics usage, the drug resistance of staphylococcus aureus is increasingly severe, and the separation rate of MRSA is also got over
Come higher.Due to clinical trials difficult and rear bad, it causes global concern.Pseudomonas aeruginosa not only causes to burn
Hurt the bacteremia of patient, and the main reason for be conduit correlation urinary tract infections (UTI) and intubation nosocomial infection.
It has natural resistance to many antibiotic, therefore is considered as multi drug resistant bacterial pathogens.Carbapenems is considered as verdigris
The first line of defence of pseudomonas infection several cases treatment, nevertheless, in the past few years, CRPA is increasing always.Bao
Infection caused by graceful acinetobacter calcoaceticus occurs mainly in respiratory system, can cause Ventilator Associated Pneumonia (VAP), bloodstream infection,
Skin and soft tissue infection, Infiltrating ductal infection, Genitourinary System Infection, meningitis etc., and not for preventing Bao Man
The effective vaccine of acinetobacter.Therefore, clinically prevent, treat and nurse infect these bacteriums patient be extremely tired
Difficult.
Reasonable employment antibacterials are the key that reduce bloodstream infection incidence and raising cure rate.It would therefore be desirable to
Fast and reliable diagnostic method accelerates Bacteria Identification in blood culture and susceptibility detection, so as to provide in time treatment appropriate and
Effective antibiotic administrative intervention.Currently, the identification and drug sensitive test of pathogenic bacteria still depend on conventional blood culture.This method
Take a long time (3 to 5 days) and need a large amount of hand labour, lacks effective detection technique, and lead to clinical biography
It catches an illness and is missed or delay diagnosis.In recent years, with the fast development of biotechnology, polymerase chain reaction (PCR), gene core
Piece, Matrix-assisted laser desorption ionization (MALDI-TOF MS) and new-generation sequencing technology obtained explore and
Using.Supplement in the Bacteria Identification and drug sensitive test of pathogen as traditional detection method, but it is limited there are still many
The problem of use.Multi-step chemical examination needed for the false negative or false positive and immunoassay process that PCR occurs during identifying
Agent all limits their application, these technologies are expensive, time-consuming and/or show hyposensitivity.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of magnetic microsphere, which provides well for the detection of microorganism
Basis.
The second object of the present invention is to provide the preparation method of magnetic microsphere described in one kind, and this method is simple and easy to do,
Good basis is provided for the extensive use of magnetic microsphere.
The third object of the present invention is to provide a kind of detection method of microorganism, micro- to judge by detection Raman signal
The type of biology, greatly shortens detection time.
The fourth object of the present invention is to provide a kind of microbial detection reagent kit, provides convenience for the detection of microorganism.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of magnetic microsphere, including microballoon ontology, the microballoon ontology carry negative electrical charge；
It is adsorbed on the PEI of the carrying positive charge of the microballoon body outer surface.
PEI is a kind of cationic polymer, has a large amount of amides and good hydrophily, PEI can be in nanometer
Particle surface adherency or assembling, the homogenization distribution of assembling easy to accomplish, to realize surface amination, provide for the detection of microorganism
Good basis.
Further, the microballoon ontology is magnetic Fe3O4Particle.
Further, the Fe3O4The partial size of particle is 200 ± 20nm.
Further, the molecular weight of the PEI is 25 ± 2kDa.
The present invention also provides the preparation methods of magnetic microsphere, comprising the following steps:
Microballoon ontology and PEI are ultrasonically treated 50-70min in a solvent, wash, obtain the magnetic microsphere.
Further, the concentration of the microballoon ontology in a solvent is 4-6g/L, and the concentration of the PEI in a solvent is 4-
6g/L。
Further, the solvent is water.
Further, the washing is carried out using distilled water.
Surface enhanced Raman scattering (SERS) detection has at low cost, easily operated, highly sensitive, quick and non-destructive testing
The advantages of, and need least sample preparation.Bacterium surface is complicated, large biological molecule, such as protein containing there are many, lipid,
Polysaccharide etc..It is difficult to carry out original position and nondestructive testing in real time by common analysis method.SERS spectra high sensitivity and
Finger-print can satisfy the requirement for quick and precisely detecting pathogenic microorganism.Early in 1989, Holter and cotton were reported for the first time
The bacterium of SERS spectrum.After 4 years, professor Magee is pointed out, can detecte pathogen completely using the dactylogram of entire organism.
Hereafter, it has been reported that the new method based on SERS detection bacterium, the i.e. fingerprint of bacterium, such as Escherichia coli, golden yellow grape
Coccus, Bacillus anthracis, bacillus subtilis, Bacillus cercus and salmonella typhimurium.Although have much about
The report of SERS enhancing substrate directly enhancing pure culture bacterium obtains the SERS fingerprint of target bacteria and carries out clustering, but faces
The research of the quick SERS detection of pathogenic bacteria is still in the exploratory stage in bed sample.Main difficulty is clinical sample complicated component, and
Common SERS enhancing substrate lacks selectively, therefore the signal of impurity understands the SERS of severe jamming target bacteria in actual sample
Signal.In addition, seldom having been reported that clinician pays special attention to this problem in quick medicament sensibility field.Refer in view of SERS
Line detects the complexity of bacterial component in the problem of pathogen and clinical sample, and the project is by by pathogen Multiple recognition
System is divided into three steps to solve these problems.The first step is to detect the finger-print of pathogen and its antibody-resistant bacterium as mark
Quasi-optical spectrum.In second step, the magnetic nanoparticle of PEI modification is used for the separation of bacterial from clinical sample, and magnetism is received
Rice grain/bacterial complex on normal blood plate and drug resistance culture plate overnight incubation to form single bacterium colony.In third
In step, the selection target single colonie from culture medium, and mixed with the high-performance SERS reinforcing material of preparation, it is then added to silicon wafer
In piece.After mixture natural air drying, detect Raman spectrum, and by the bacterium spectrum of acquisition and we measure in the first step
Standard spectrum is compared, to determine whether it is corresponding pathogenic bacteria.Obviously, the bacterium colony grown on medicaments insensitive culture plate
It can only be corresponding drug-resistant bacteria.Bacterium colony is mixed with SERS reinforcing material (Ag NPs) with detect spectrum and with standard spectrum ratio
Compared with to determine whether pathogen is resistant.SERS finger-print and medicaments insensitive culture plate can with dual determining pathogenic bacteria and its
Antibody-resistant bacterium.
A kind of detection method of microorganism provided by the invention, comprising the following steps:
Above-mentioned magnetic microsphere adsorbs object to be detected, detects Raman signal, is judged according to the peak value of Raman signal to be detected
The microbe species of object；
The object to be detected is the sample containing the microorganism for carrying negative electrical charge.
Further, the object to be detected is pathogen.
Further, the pathogen includes in staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa
It is any one or more.
Further, the sample to be tested is blood sample.
Magnetic microsphere i.e. provided by the invention can effectively in adsorption sample microorganism, reduce the interference of other impurities.
In actual operation, after sample mixes incubation a period of time with magnetic microsphere, by the magnetic absorption outside test tube, make
The magnetic microsphere and magnet absorption of microorganism must be carried, other impurities is can remove in this way, is then washed as needed, into
One step removes impurity.
Currently, clinical labororatory's program for detecting blood samples of patients sample is that Blood culture bottle is put into automatic blood training first
It supports instrument and carries out Bacteria Culture.After the positive findings (1-2 days) of bacterial multiplication to instrument, Blood culture bottle is taken out, is then carried out flat
Plate bacterium colony culture, and Bacteria Identification and drug susceptibility detection are carried out after one day.Whole process takes around 3-5 days.
And method of the invention is when receiving Blood culture bottle by magnetic microsphere such as Fe3O4@PEI is put into Blood culture bottle, is caught
It obtains and is enriched with the bacterium in blood, then by the Fe of collection3O4@PEI@bacterial complex is coated in ordinary flat and medicaments insensitive
Plate, 37 DEG C of overnight incubations, selection target pathogenic bacteria carry out Raman spectrum detection, and are compared with the standard spectrum established before
Compared with.By combining spectrum with the growth of the chemometrics application of OPLS-DA and common plate and medicaments insensitive plate, realize
The identification of target pathogenic bacteria, to clinically save the process of Enrichment of bacteria in flashing lightning magnetic field detector, and whole process
It has only used 1-2 days.
Fe3O4@PEI nanoparticle has the ability of wide spectrum capture bacterium, because the positive charge on its surface passes through electrostatic attraction
It interacts with the negative electrical charge on the cell wall of Gram-positive and gramnegative bacterium, to obtain good bacterium capture
Efficiency (staphylococcus aureus 97.87%, Acinetobacter bauamnnii 80.57%, pseudomonas aeruginosa 97.25%).
Further, the object to be detected is pathogen.
Further, the pathogen includes in staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa
It is any one or more.
Further, the sample to be tested is blood sample.
Further, the sample to be tested is mixed with magnetic microsphere, so that magnetic microsphere adsorbs object to be detected.
Further, it is incubated for, is jiggled during being incubated for, incubation time 15-20min after mixing.
The present invention has studied the Fe of different incubation times3O4The bacterium capture rate of@PEI.Fe3O4@PEI effectively captures golden yellow
The incubation program of color staphylococcus, Acinetobacter bauamnnii and pseudomonas aeruginosa only spends 15 minutes.
Further, target thallus content is 10 in the sample to be tested1CFU/mL-103CFU/mL；
Further, 104The weight of magnetic microsphere needed for CFU/mL thallus is 0.1mg.
Invention also contemplates that Fe3O4The influence of concentration of the@PEI in bacterium captures.Use the Fe of different volumes3O4@
PEI (2,4,6,8 and 10 μ L) captures identical staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa sample in order
Product (1mL, 104CFU·mL-1), with Fe3O4The dosage of@PEI increases, and the capture rate of bacterium increases, and obtains bacterium and captures institute
The Fe of the optimal dose needed3O4@PEI。
Further, after magnetic microsphere absorption object to be detected is mixed with enhancing semiochemicals, drop on silicon draw by measurement
Graceful signal.
Strength difference between Raman signal peak is mainly caused by the content of nucleic acid, amino acid and lipid.Nucleic acid, amino acid
Play a crucial role in biochemical reaction with lipid, for example, bacteria cell wall synthesis process and structure composition and bacterium generation
It thanks.Therefore, variation relevant to bacterial resistance can be reflected by the spectral intensity and difference in migration of bacterium.
The cell wall of staphylococcus aureus peptide glycan rich in and a small amount of teichoic acid, and peptide glycan is netted
Structure is more more dense than the reticular structure of gramnegative bacterium.However, Acinetobacter bauamnnii and pseudomonas aeruginosa have cell
Wall peptidoglycan layer, the degree of cross linking is poor, and lipid content is high, and there are significant differences for the cell wall structure and metabolism between three kinds of bacterial strains.Cause
This, lipid (1458cm-1), protein (654cm-1,637cm-1,958cm-1) and carbohydrate (731cm-1,1132cm-1)
Content is also obvious there is also significant difference, the difference of corresponding spectrum.In 654cm-1,731cm-1And 1458cm-1The SERS item at place
Band is attributed to tyrosine, guanine, adenine, DNA and saturated fatty acid, is shown in MSSA higher hundred than in MRSA
Divide and compares signal.MRSA has multiple resistance mechanism, and the drug resistance of beta-lactam antibiotic is mainly due to MRC chromosome mec
The presence of A gene, codified binding protein is to synthesize new specific penicillin P BP2a.When beta-lactam antibiotic is made
Used time, normal PBPs is in conjunction with antibiotic and loses the synthesis of cell wall, and PBP2a is not inhibited, and replaces normal PBP work
Make, complete the synthesis of cell wall, make bacteria living and becomes antibody-resistant bacterium.MRSA can also generate beta-lactam by plasmid
Enzyme, slowly inactivation shows β-lactamide antibiotic of drug resistance.In the synthesis process of MRSA cell wall, glucose is bacterium
The good sources of carbon of growth and energy source, it is also necessary to synthetic proteins matter, so that corresponding spectral intensity variation occur in spectrum.
Coacetylase, acetyl coenzyme A (724cm-1) SERS band in CRAB be higher than CSAB.Acetyl coenzyme A be energetic supersession it is important in
Between metabolite, be the key substance in bacterium energetic supersession.It can be conducive to release energy with sintetics, be life
It uses.CRAB mainly includes the generation of beta-lactamase, Active efflux-pump system to the resistance mechanism of beta-lactam antibiotic
Overexpression, the change of penicillin binding protein and the reduction of outer membrane permeability.These variations need a large amount of energy, also can
The main reason for composition of influence CRAB cell wall structure, this is SERS spectra difference between CRAB and CSAB.Due to di(2-ethylhexyl)phosphate
Ester, deoxyribose, adenine, poly- adenine, tryptophan, adenine and guanine are in 911cm-1,1321cm-1And 1351cm-1Place
SERS band different signals is shown between CRPA and CSPA.Glucose metabolism and ammonia metabolism lead to amino acid, sugar and
The difference of other metabolins, and the variation of purine, pyrimidine and other substances is related with the amplification of bacterium amplifying nucleic acid.The drug resistance of CRPA
Mechanism is complicated, mainly includes five kinds of mechanism, i.e. the variation of outer membrane permeability, the reduction or disappearance of drug target, inactivator or
The generation of modification enzyme, the formation of Active efflux-pump system and biomembrane.These mechanism will lead to CRPA cell wall protein type
Variation, and significant impact, the main reason for this is SERS spectra difference between CRPA and CSPA, are generated to cell wall constituent.Cause
This, the relationship understood between microbial metabolism and bacterial resistance mechanisms generation extremely closes identification different bacterium and its tolerant bacteria
It is important.
The difference of X is divided into two parts according to the difference of Y by OPLS-DA.First part's expression difference relevant to Y, second
Part indicates the difference unrelated with Y (orthogonal vertical).OPLS-DA can distinguish the difference between the two parts.It is intended to control
System and filtering X in directly intersect with Y or independently of Y variation.In this way, OPLS-DA can better discriminate between the difference between group, mention
The validity and analysis ability of high model.The OPLS-DA shot chart for the four kinds of models established in this research is shown at same group
In cluster, and there are apparent separation trends between different group.We also estimate mould by carrying out 10 times of cross validations
The classification effectiveness of type.The minimum training of model and the accuracy of test set are respectively greater than 98% and 96%.These data show that
The feasibility of different pathogens disaggregated model is established using OPLS-DA algorithm.
In addition, detection bacterium bacterial strain is sequenced by two generations, successful identification of the present invention bacterium is demonstrated.It is visually observing
Under, the SPECTRAL DIVERSITY between sensitive bacteria bacterial strain of the same race and antibody-resistant bacterium is unobvious.It is being detected to improve SERS finger-print
Accuracy in antibody-resistant bacterium, the present invention implement a kind of based on common/drug sensitive plate single colonie growth and SERS finger-print ratio
Compared with scheme.In this study, the bacterium of Magnetic Isolation is cultivated simultaneously on the common and plate of medicaments insensitive.Sensitive bacterial meeting
Grow bacterium colony in ordinary flat, but not grown on medicaments insensitive plate, and antibody-resistant bacterium can in medicaments insensitive plate and
Single colonie is grown in ordinary flat, then SERS dactylogram combination OPLS-DA statistical analysis technique is for further confirming that it is
No is a kind of antibiotic resistant pathogens.By drug susceptibility screening and the dual identification of SERS finger-print combination OPLS-DA,
The antibody-resistant bacterium that can more accurately detect pathogen, without extending detection time.
The present invention constructs a Multiple recognition system, the magnetic nano-particle quick separating pathogen modified using PEI,
Then the single bacterium colony for being further cultured on common/medicaments insensitive plate, and being grown using SERS dactylogram combination OPLS-DA simultaneously,
To accurately identify pathogen and its antibody-resistant bacterium.Entire detection system does not need complicated sample pretreatment, easy to operate, is not necessarily to
Professional operation, advantage of lower cost.The successful implementation of the detection scheme will detect and susceptibility for the quick of clinical important pathogenic bacteria
Test provides new thinking and solution.
Further, drug-fast bacteria and non-drug-fast bacteria are distinguished using orthogonal partial least squares discriminant analysis.
Further, the detection of drug-fast bacteria and non-drug-fast bacteria further include on susceptibility culture plate cultivate it is resistance to be confirmed whether
Medicine.
Further, before detecting Raman signal further include the steps that enhancing semiochemicals are added.
Further, the enhancing semiochemicals include any one of nano-Ag particles, colloidal gold, Jin Heyin shell.
The solution of Ag nano particle has absorption maximum and relatively narrow half-breadth at 407nm, shows that particle is uniform
And there is good dispersibility.The present invention has studied Ag NP (silver nano-grain) and adsorbs on object to be detected in magnetic microsphere
Distribution situation finds that the distribution of Ag NP is relatively uniform.
In order to prove Ag NP to the enhancing ability of pathogenic bacteria SERS signal, the present invention compares addition and does not add Ag NP
SERS signal intensity.It was found that addition Ag NP can dramatically increase the intensity at many peaks, show strong between Ag NP and pathogen
Interaction.Therefore, SERS enhancing basis of the silver sol as pathogen is selected in the present invention.Certainly, enhance semiochemicals
It can also be colloidal gold, Jin Heyin shell etc..
The present invention also provides a kind of microbial detection reagent kits, including the magnetic microsphere.
Further, the detection kit further includes any one of silicon wafer, enhancing semiochemicals, magnet or more
Kind.
Further, the enhancing semiochemicals include any one of nano-Ag particles, colloidal gold, Jin Heyin shell.
The present invention provides microbial detection reagent kit, provides convenience for the detection of microorganism.
Compared with prior art, the invention has the benefit that
(1) magnetic microsphere provided by the invention, PEI can be adhered to or be assembled, assembling easy to accomplish in nanoparticle surface
Homogenization distribution, to realize surface amination, provide good basis for the detection of microorganism.
(2) microorganism that magnetic microsphere provided by the invention can effectively in adsorption sample, high specificity adsorb more tight
It is close, reduce the interference of other impurities.
(3) detection method of microorganism provided by the invention greatly shortens detection time, and method is convenient reliable, accuracy rate
It is 96% or more, provides a kind of novel, quick detection mode for the diagnosing and treating of clinical blood infection.
(4) present invention provides microbial detection reagent kit, provides convenience for the detection of microorganism.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 be the present embodiments relate to detection schematic diagram；
Fig. 2 is Fe in the embodiment of the present invention 13O4The characterization correlation figure of 3 property of@PEI；
Fig. 3 is Fe in the embodiment of the present invention 13O4Capture rate correlation figure of the@PEI to bacterium；
Fig. 4 is the antimicrobial efficiency figure of various concentration antibiotic in the embodiment of the present invention 2；
Fig. 5 is the related figure of SERS detection of 2 bacterium of the embodiment of the present invention；
Fig. 6 is that the SERS of 2 bacterium of the embodiment of the present invention detects multivariate statistical analysis figure.
Specific embodiment
Working principle for Bacteria Identification and the dual identification SERS biosensor of drug-resistant test is as shown in Figure 1B.
Figure 1B shows while identifying the operation sequence of microorganism and microorganism medicine sensitive, by SERS biosensor to golden yellow Portugal
The resistance of grape coccus, Acinetobacter bauamnnii and pseudomonas aeruginosa.
Particular content is as follows:
Firstly, the high performance material of preparation pathogenicbacteria separation and fingerprint signal enhancing suitable for blood sample, establishes mark
The bacterium SERS finger-print of standardization；
Secondly, the magnetic nano-particle of PEI modification is used for the separation of bacterial from blood sample, and pass through blood plate and anti-medicine
Property culture plate culture；
Finally, carry out precise Identification staphylococcus aureus using the SERS fingerprint from single colonie, Acinetobacter bauamnnii and
Pseudomonas aeruginosa and resistant strain (MRSA, CRAB and CRPA).This method provided by the invention is clinical blood infection
Diagnosing and treating provides a kind of novel, quick detection mode.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
1, chemicals and material
Polyethyleneimine branching (PEI, MW 25kDa), silver nitrate (AgNO3) obtained from Sigma-Aldrich.From
Sinopharm Chemical Reagent Co. buys iron chloride (III) hexahydrate (FeCl3·6H2O), polyethylene glycol
(PEG6000), trisodium citrate (TSC), ethylene glycol (EG) and anhydrous sodium acetate (NaAc).Other reagents are tried from Shanghai chemistry
Agent Co., Ltd (China).All chemicals are analysis level.
Aqueous solution is prepared with Millipore ultrapure water, the purifying in Milli-Q system (18.2M Ω cm-1).It is cured from Xuzhou
Staphylococcus aureus, the clinical separation strain of pseudomonas aeruginosa and Acinetobacter bauamnnii are collected by affiliated hospital, university, section.According to
Clinical and laboratory standard research institute breakpoint standard (CLSI M100, S27) defines methicillin (staphylococcus aureus) and sub-
Amine trains southern (pseudomonas aeruginosa, Acinetobacter bauamnnii) resistance.All participants both provide informed consent.All means are pressed
Implement according to the guilding principle of Xuzhou Ethics Committee, affiliated hospital, medical university approval.
2, it characterizes
2600 spectrometer of Shimadzu records ultraviolet-visible spectrum.Use the dynamic of Brookhaven Zeta PALS instrument
State light scatters the zeta current potential that (DLS) measures all nano particles.Pass through JEOL JEM-2010F microscope and Hitachi H-
7650TEM obtains high resolution transmission electron microscope (HRTEM) image and TEM image under the acceleration voltage of 200kV respectively.
Study the magnetism of the MNP of preparation at 300k by superconducting quantum interference device magnetometer (SQUID, MPMSXL-7).Raman light
Spectrum records on portable Raman system B&W Tek, i-Raman Plus BWS465-785H spectrometer.All samples by
The excitation of 785nm laser, power 25mW, the total acquisition time of each SERS spectra are 20s.There is provided measurement spectrum be used for than
Compared with then Calibration Base Line.
3, prepared by bacteria samples
Use the concentration of common colony counting method measurement bacterium.In short, by staphylococcus aureus, P. aeruginosa
Bacterium and Acinetobacter bauamnnii in Luria-Bertani (LB) culture medium in 37 DEG C culture 3-5 hours.Then, dilute with culture medium
Release 0.1mL bacterial cultures 1 × 105It is a, and be laid on agar plate, the overnight incubation at 37 DEG C.Hereafter, bacterium is obtained
Fall the quantity to form unit (CFU).
4、Fe3O4The preparation of@PEI
As shown in Figure 1A, Fe is synthesized by two steps3O4@PEI microballoon.Firstly, being synthesized using improved solvent thermal reaction
Magnetic Fe3O 4Particle (200nm).In general, by 2.7g FeCl3·6H2O is dissolved in 80mL ethylene glycol, and magnetic agitation 30 is divided
Clock states addition 5.4g NaAc and 2g PEG6000 in solution then up, and stirs until reactant sufficiently dissolves.Then, will
Mixture is transferred in the autoclave (100mL capacity) of teflon lined, and is heated mixture 6 hours at 210 DEG C.It will
Product is used deionized water and ethanol washing three times respectively, and is collected with magnet.Final product is dried in vacuo 6 hours at 60 DEG C.
Secondly, preparing Fe under ultrasound condition3O4@PEI microballoon, wherein the cationic self assembly of PEI is in Fe3O4On the surface of particle.
In brief, the 500mg Fe3O4 particle prepared is dispersed in PEI solution (0.5g/100mL) 1 hour under ultrasonic treatment,
PEI during this period gradually self assembly in Fe3O4On particle.Then by Fe3O4@PEI microballoon Magneto separate and with Milli-Q water washing
Five times to wipe excessive PEI.
The feature (Fig. 2, A-E) of synthetic product is obtained by TEM image.Hydrophily PEI can quickly be assembled in Fe3O4It is micro-
On the surface of ball, constitute thin layer (Fig. 2A).PEI layers (Fig. 2 B) are further confirmed by HRTEM image, and it is super to be shown in half an hour
PEI's with a thickness of about 2.7nm after sonication.
In order to further verify Fe3O4@PEI studies the Fe to be formed by TEM image to the capture ability of bacterium3O4@
PEI/ bacterial complex.Fig. 2 C-E shows Fe3O4@PEI effectively can capture staphylococcus aureus, Bao Man not from solution
Lever bacterium and pseudomonas aeruginosa.Fig. 2 C is the microscopic observation figure of staphylococcus aureus clustering phenomena；Fig. 2 D is that Bao Man is motionless
The microscopic observation figure of bacillus clustering phenomena；Fig. 2 E is the microscopic observation figure of pseudomonas aeruginosa clustering phenomena.
Fig. 2 F shows when PEI is modified, Fe3O4The zeta potential of particle increases to+41.1mV from -34.9mV.Golden yellow Portugal
The zeta current potential of grape coccus, Acinetobacter bauamnnii and pseudomonas aeruginosa is respectively -50.4mV, -23.9mV and -34.6mV.?
Fe3O4After modifying PEI on microballoon, Fe3O4The zeta potential of@PEI becomes strong positive (+41.1mV) because on surface there are many sun from
Sub- amine groups.Fe3O4The positive surface charge of@PEI microballoon provides the effective tool separated by electrostatic interaction.Work as application
When external magnets, Fe3O4@PEI microballoon can be separated from solution (1mL) in 1 minute.Which reflects Fe3O4@PEI microballoon
Potentiality for fast enriching target bacteria.
5, enhance nanoparticle
Use the spare collargol for making SERS enhancing matrix of reduction of sodium citrate legal system.Firstly, weighing 33.72mg AgNO3
And it is added into 200mL distilled water until boiling.Then the trisodium citrate that 8mL volume fraction is 1% is once added
It states in solution and is stirred with 650rpm.After 45 minutes, stop heating.During continuously stirring, solution is gradually cooling to room temperature,
Then deionized water is added into solution until volume reaches 200mL.It is from the reactions above by collargol (1mL) using sample rifle
Liquid relief of uniting is managed to EP, is then centrifuged 7 minutes with 7000rpm.After removing supernatant, remaining sediment is resuspended in 100 μ
In L distilled water for future use.
6, the magnetic capture and SERS measurement of bacterium
Preparing concentration range is 101CFU/mL to 103The bacteria samples of CFU/mL, then to 10 μ g/mL are added in every group
Fe3O4@PEI, and mixture incubation is jiggled on a shaker.After twenty minutes, using external magnets by Fe3O4@PEI@is thin
Bacterium compound is adsorbed onto EP bottom of the tube, discards supernatant liquid, is then rinsed with PBST twice to remove interfering substance.Later, successively
100 μ L sterile waters, the Fe that will be obtained is added3O4@PEI@bacterial complex Columbia blood culture medium (bioM'erieux,
La Balme-les-Grottes, France) upper 37 DEG C be incubated overnight.The bacterium colony that selection is collected using aseptic inoculation ring, is laid equal stress on
It is suspended from 10 μ L sterile waters.The Ag NP that 10 μ L concentration are 10 times is added (about 1 × 10 in bacterial suspension8CFU/mL).Sufficiently
After mixing, mixture drop is measured to their Raman signal on clean silicon wafer.
7、Fe3O4Capture rate of the@PEI to bacterium
Fe3O4@PEI captures the ability of different bacterium from blood sample.Different bacterium includes staphylococcus aureus, Bao
Graceful acinetobacter calcoaceticus and pseudomonas aeruginosa.Blood agar plate shows most of bacteriums by Fe3O4@PEI capture, calculates in supernatant
Remaining bacterium calculates capture rate (Fig. 3 A).Show Fe by counting3O4@PEI is to staphylococcus aureus, Bao Man not lever
The capture rate of bacterium and pseudomonas aeruginosa is respectively 97.87%, 80.57% and 97.25%, can rapid enrichment and separation bacterium
(Fig. 3 B-D).Fig. 3 A indicates blood agar plate display capture Fe3O4After@PEI in supernatant remaining bacteria amount, with unmodified
Fe3O4As control.Fig. 3 B-D is respectively indicated to staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa
Fe3O4The respective capture rate of@PEI.*P < 0.05,**P < 0.01,***P<0.001。
Fe of the present invention also to different incubation times3O4The bacterium capture rate of@PEI is tested.Fe3O4@PEI is effective
The incubation program of capture staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa only spends 15 minutes.
The present inventor also contemplates Fe3O4The influence of concentration of the@PEI in bacterium captures.Use the Fe of different volumes3O4@
PEI (2,4,6,8 and 10 μ L) captures identical staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa sample in order
Product (1mL, 104CFU·mL-1).It was found that with Fe3O4The dosage of@PEI increases, and the capture rate of bacterium increases, and works as
Fe3O4The volume of@PEI reaches balance when being 10 μ L.Therefore, Fe3O4@PEI (10mgmL is selected-1) amount be 10 μ L, and
The incubation time of Fe3O4@PEI is 15min in this study.
Embodiment 2
1, antibiotic susceptibility test
By agar dilution testing inspection to the sensibility or drug resistance of drug or drug categories, Mueller- is mainly used
The sensitivity testing to antibacterials of Hinton agar (MHA).The antimicrobial of 10 × concentration is prepared with 1280 μ g/mL, and even
It is continuous to be diluted to required ultimate density twice.In order to prepare intermediate diluent, the convenient formula used is C1*V1=C2*
V2, wherein C1 is the concentration (usually 1280 μ g/mL or higher) of antimicrobial stoste, and V1 is needed for generating intermediate concentration
Unknown volume, C2 are intermediate concentrations, and V2 is the volume for the intermediate stock solution that we need.The solvent and diluent of Imipenem
It is phosphate buffer (pH7.2,0.01mol/L).The solvent and diluent of oxacillin are water.With Mueller-Hinton fine jade
Rouge dilutes 1:10 to obtain the ultimate density of antibiotics sensitivity agar plate.Use the μ of the MHA containing 4%NaCl and 1,2,4,8
G/mL oxacillin detects MRSA, in 35 DEG C of ± 2 DEG C of overnight incubations.In addition, we use the μ of MHA and 1,2,4,8 g/mL imines
South is trained to detect CRPA and CRAB.
Fig. 4 shows the antimicrobial efficiency (%) of various concentration antibiotic, and Fig. 4 A is for MRSA and MSSA, methicillin antibiosis
Plain concentration is respectively 0,1,2 and 4 μ g/mL；For Fig. 4 B for CRAB and CSAB, Imipenem concentration is respectively 0,1,2 and 4 μ g/mL；
For Fig. 4 C for CRPA and CSPA, Imipenem concentration is respectively 0,1,2 and 4 μ g/mL；Error line indicates the reality carried out in triplicate
The standard deviation tested.
From, as can be seen that MRSA remains to grow on medicaments insensitive plate, MSSA is in the medicine that antibiotic concentration is 2 μ g in Fig. 4 A
It is not grown on object sensitive plate；As can be seen that CRAB still can be in each concentration growth of Imipenem, CSAB from Fig. 4 B
It is not grown on the drug sensitive plate that antibiotic concentration is 2 μ g/mL；Can be seen that CRPA from Fig. 4 C still can be each in Imipenem
It is grown under concentration conditions, and CSPA is not grown on the drug sensitive plate that antibiotic concentration is 2 μ g/mL.
2, Raman spectrum detects
Raman spectrum records on portable Raman system B&W Tek, i-Raman Plus BWS465-785H spectrometer.
All samples are excited by 785nm laser, power 25mW, and the total acquisition time of each SERS spectra is 20s.Measurement is provided
Spectrum for comparing, then Calibration Base Line.
The 70 kinds of encountered pathogenic bacterias infected from clinical blood, 13 plants of MRSA, 10 plants of MSSA, 17 plants of CRAB, 11 plants of CSAB, 9
The SERS signal of bacterium is obtained in CRPA and 10 plant of CSPA based on Ag NPs of strain.Fig. 5 A shows MRSA, MSSA, CRAB,
The average SERS spectra of CSAB, CRPA and CSPA, have directly displayed the difference of the SERS spectra of bacterium bacterial strain.In Fig. 5 A, MRSA
Average SERS intensity (black line, n=13), MSSA (red line, n=10), CRAB (blue line, n=17), CSAB (pink colour line, n=
11), CRPA (green line, n=9) and CSPA (dark blue colo(u)r streak, n=10).It is received under the laser power of 785nm excitation (25mW, 10s)
Collect spectrum, subtracts baseline and to carry out spectrum smooth.Shadow region represents standard deviation.
Following main peak: 592,637,654,731,791,886,958,1132,1204,1249,1328,1458 Hes
1657cm-1, mainly by glucosides annular strain, l-tyrosine, DNA, lactose, tyrosine, guanine, poly- adenine, cytimidine urinates phonetic
Pyridine, amide III, protein, D-MANNOSE, phenylalanine, lipid, saturated fatty acid, amine-galactose, amide I, in golden yellow
It is different in staphylococcus, Acinetobacter bauamnnii and pseudomonas aeruginosa.In staphylococcus aureus, respectively by adenine, poly- gland
731,958, the 1328 and 1458cm that purine, DNA, protein, saturated fatty acid rise-1Intensity be above other two bacterial strains.
592,637,886,1132,1204 and 1657cm-1The band at place is attributed to l-tyrosine, glucosides annular strain, amino gala respectively
Sugar, D-MANNOSE, phenylalanine, lactose, protein, amide III, amide I, compared with other two kinds of bacterial strains, Bao Man not lever
The content of bacterium is higher.
The present invention also compares the SERS difference of bacteria samples between drug-resistant bacteria and medicaments insensitive bacterium.Drug-resistant bacteria and
The Raman peaks of medicaments insensitive bacterium are relatively similar.Compared with MRSA, stronger peak includes 654cm in MSSA-1(tyrosine, bird
Purine), 731cm-1(poly- adenine, DNA, adenine) and 1458cm-1The peak of (saturated fatty acid).Peak value ratio in CSAB
CRAB becomes apparent from, including 592cm-1(COC glucosides annular strain), 637cm-1(l-tyrosine, lactose), 1132cm-1(D- sweet dew
Sugar), 1204cm-1(tyrosine, phenylalanine, protein, amide III) and 1657cm-1(amide I).And and 724cm-1CSAB
(coacetylase, acetyl coenzyme A) is compared, and the peak in CRAB is stronger.
Finally, comparing the SERS spectra between CRPA and CSPA.We have found that SERS signal intensity is in 911,1321 and
1351cm-1The difference at place relates generally to di-phosphate ester, deoxyribose, adenine, poly- adenine, tryptophan, adenine, bird
Purine.
In order to further appreciate that the difference at the peak bacterium SERS, table 1 lists main Raman peak position and the vibration of different molecular
Dynamic model formula and main component.
The difference at 1 peak bacterium SERS of table
3, Raman spectrum multivariate statistical analysis
Raman spectrum data analysis is statisticallyd analyze to carry out in SIMCA 14.1 (Umetrics, Umea, Sweden).It is original
Data and normalized number are zoomed in and out by unit variance accordingly centered on average value.Orthogonal partial least squares discriminant analysis
(OPLS-DA) it is supervision statistical method for discriminant analysis.This method is by establishing metabolin expression and sample classification
Between relational model realize the prediction of sample classification.In the parameter evaluation obtained by model, R2X and R2Y are respectively indicated
The explanation rate of the X and Y matrix model of foundation, Q2 indicate the predictive ability of model.Theoretically, the value of R2 and Q2 is closer to 1,
Model is better, and the fitting precision of model is lower.In addition, assessing OPLS-DA model prediction unknown sample by 10 times of cross validations
The ability of classification.That is, data set is divided into 10 parts, use 9 parts as training data in turn, 1 part is as survey
Examination data are tested.Test all obtains accuracy or error rate every time.Use 10 accuracy of result or putting down for error rate
Mean value carrys out the accuracy of algorithm for estimating.It is often necessary to 10 times cross validation 10 times, calculate average value then with the standard of estimating algorithm
True property.
From 23 plants of staphylococcus aureuses (13 plants of MRSA, 10 plants of MSSA), and 28 plants of Acinetobacter bauamnniis (17 plants of CRAB, 11
Strain CSAB), the SERS spectra of the bacterium obtained in 28 Pseudomonas aeruginosa strains (compares 9 plants CRPA and 10 plant using OPLS-DA
CSPA).Fig. 5 B shows the visible trend of three kinds of bacteriums, and then we distinguish these three types and secondary using OPLS-DA
To distinguish their drug-resistant bacteria respectively.Fig. 6 A shows the sample for the three types being completely separated from each other, and specifically joins
Number is R2X (cum)=0.703, R2Y (cum)=0.914 and Q2 (cum)=0.912.Based on 10 times of cross validations, OPLS-
The training set of DA and the Average Accuracy of test set are respectively 99.92% and 98.34%.We also compare drug-resistant bacteria and medicine
Difference between object sensitive bacterial.Between MRSA and MSSA, Fig. 6 B shows being clearly separated according to score scatter plot.Specifically
Parameter is: R2X (cum)=0.823, R2Y (cum)=0.892, Q2 (cum)=0.792.The result of these three parameters is both greater than
0.7, show that the model is established well and has good predictive ability.We have also carried out 10 cross validation realities
It tests, the accuracy classified with assessment models.The training of bat and test set are respectively 98.15% and 96.25%.?
Find that they are slightly overlapped (Fig. 6 C) in shot chart between CRAB and CSAB using OPLS-DA.Model parameter is R2X
(cum)=0.781, R2Y (cum)=0.866, Q2 (cum)=0.833.It is trained and test set flat by ten times of cross validations
Equal accuracy rate is respectively 99.21% and 98.39%, is shown functional.Finally, we compare between CRPA and CSPA
OPLS-DA score curve.Fig. 6 D shows that two kinds of bacterium bacterial strains are separated with minimum overlay.Model parameter is R2X (cum)=0.797,
R2Y (cum)=0.793, Q2 (cum)=0.724, shows good predictive ability.We also use ten times of cross validations
The classification capacity of assessment models.Trained and test set Average Accuracy is respectively 98.28% and 96.82%.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of magnetic microsphere, which is characterized in that including microballoon ontology, the microballoon ontology carries negative electrical charge；
It is adsorbed on the PEI of the carrying positive charge of the microballoon body outer surface；
Further, the microballoon ontology is magnetic Fe3O4Particle；
Further, the Fe3O4The partial size of particle is 200 ± 20nm；
Further, the molecular weight of the PEI is 25 ± 2kDa.
2. the preparation method of magnetic microsphere described in claim 1, which comprises the following steps:
Microballoon ontology and PEI are ultrasonically treated 50-70min in a solvent, wash, obtain the magnetic microsphere；
Further, the concentration of the microballoon ontology in a solvent is 4-6g/L, and the concentration of the PEI in a solvent is 4-6g/
L；
Further, the solvent is water；
Further, the washing is carried out using distilled water.
3. a kind of detection method of microorganism, which comprises the following steps:
Magnetic microsphere described in claim 1 adsorbs object to be detected, detects Raman signal, according to the peak value of Raman signal judge to
The microbe species of detectable substance；
The object to be detected is the sample containing the microorganism for carrying negative electrical charge.
4. detection method according to claim 3, which is characterized in that the object to be detected is pathogen；
Further, the pathogen includes any in staphylococcus aureus, Acinetobacter bauamnnii and pseudomonas aeruginosa
Kind is a variety of；
Further, the sample to be tested is blood sample.
5. detection method according to claim 3, which is characterized in that the sample to be tested is mixed with magnetic microsphere, is made
It obtains magnetic microsphere and adsorbs object to be detected；
Further, it is incubated for, is jiggled during being incubated for, incubation time 15-20min after mixing.
6. detection method according to claim 3, which is characterized in that target thallus content is in the sample to be tested
101CFU/mL-103CFU/mL；
Further, 104The weight of magnetic microsphere needed for CFU thallus is 0.1mg.
7. detection method according to claim 3, which is characterized in that detect Raman signal on silicon.
8. detection method according to claim 3, which is characterized in that distinguished using orthogonal partial least squares discriminant analysis resistance to
Medicine bacterium and non-drug-fast bacteria；
Further, the detection of drug-fast bacteria and non-drug-fast bacteria further includes cultivating on susceptibility culture plate to be confirmed whether drug resistance.
9. according to the described in any item detection methods of claim 3-8, which is characterized in that further include being added before detection Raman signal
The step of enhancing semiochemicals；
Further, the enhancing semiochemicals include any one of nano-Ag particles, colloidal gold, Jin Heyin shell.
10. a kind of microbial detection reagent kit, which is characterized in that including magnetic microsphere described in claim 1；
Further, the detection kit further includes that silicon wafer, enhancing semiochemicals, magnet are any one or more of；
Further, the enhancing semiochemicals include any one of nano-Ag particles, colloidal gold, Jin Heyin shell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114214225A (en) * | 2021-11-28 | 2022-03-22 | 中国人民解放军军事科学院军事医学研究院 | Method for enriching holofast secreted by brevibacterium crescentum |
| CN114535593A (en) * | 2021-11-26 | 2022-05-27 | 河南农业大学 | Preparation method and application of AgNPs @ SASP substrate material |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344117A (en) * | 2010-08-06 | 2012-02-08 | 同济大学附属上海市肺科医院 | Method for preparing composite nano microsphere for enriching lung cancer cells |
| CN102460172A (en) * | 2009-05-07 | 2012-05-16 | 生物梅里埃有限公司 | Method for Antimicrobial Resistance Determination |
| CN102502879A (en) * | 2011-11-02 | 2012-06-20 | 华东师范大学 | A kind of Fe3O4 nano microsphere and preparation method thereof |
| CN105738340A (en) * | 2015-11-05 | 2016-07-06 | 新疆大学 | Quick detection method of lavender essential oil categories on the basis of Fourier-Raman spectrum |
| CN106323935A (en) * | 2015-07-06 | 2017-01-11 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Magnetic composite SERS substrate with core-shell-satellite three dimensional structures and preparation method thereof |
| CN106997799A (en) * | 2016-01-22 | 2017-08-01 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of preparation method and its SERS application of high-performance gold shell magnetic microballoon |
| CN107505304A (en) * | 2017-07-26 | 2017-12-22 | 中国人民解放军第二军医大学 | A kind of quick method for judging drug-resistant type Candida albicans |
| CN107586823A (en) * | 2017-10-10 | 2018-01-16 | 徐州医科大学 | The rapid identification method of Carbapenems drug susceptibility based on Raman spectroscopy |
| CN108060178A (en) * | 2017-12-29 | 2018-05-22 | 佛山科学技术学院 | A kind of magnetic Nano genophore of nucleocapsid and preparation method thereof |
-
2019
- 2019-01-18 CN CN201910049833.XA patent/CN109781702A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102460172A (en) * | 2009-05-07 | 2012-05-16 | 生物梅里埃有限公司 | Method for Antimicrobial Resistance Determination |
| CN102344117A (en) * | 2010-08-06 | 2012-02-08 | 同济大学附属上海市肺科医院 | Method for preparing composite nano microsphere for enriching lung cancer cells |
| CN102502879A (en) * | 2011-11-02 | 2012-06-20 | 华东师范大学 | A kind of Fe3O4 nano microsphere and preparation method thereof |
| CN106323935A (en) * | 2015-07-06 | 2017-01-11 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Magnetic composite SERS substrate with core-shell-satellite three dimensional structures and preparation method thereof |
| CN105738340A (en) * | 2015-11-05 | 2016-07-06 | 新疆大学 | Quick detection method of lavender essential oil categories on the basis of Fourier-Raman spectrum |
| CN106997799A (en) * | 2016-01-22 | 2017-08-01 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of preparation method and its SERS application of high-performance gold shell magnetic microballoon |
| CN107505304A (en) * | 2017-07-26 | 2017-12-22 | 中国人民解放军第二军医大学 | A kind of quick method for judging drug-resistant type Candida albicans |
| CN107586823A (en) * | 2017-10-10 | 2018-01-16 | 徐州医科大学 | The rapid identification method of Carbapenems drug susceptibility based on Raman spectroscopy |
| CN108060178A (en) * | 2017-12-29 | 2018-05-22 | 佛山科学技术学院 | A kind of magnetic Nano genophore of nucleocapsid and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| CHONGWEN WANG等: "A rapid SERS method for label-free bacteria detection using polyethylenimine-modified Au-coated magnetic microspheres and Au@Ag nanoparticles", 《ANALYST》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114535593A (en) * | 2021-11-26 | 2022-05-27 | 河南农业大学 | Preparation method and application of AgNPs @ SASP substrate material |
| CN114535593B (en) * | 2021-11-26 | 2023-03-31 | 河南农业大学 | Preparation method and application of AgNPs @ SASP base material |
| CN114214225A (en) * | 2021-11-28 | 2022-03-22 | 中国人民解放军军事科学院军事医学研究院 | Method for enriching holofast secreted by brevibacterium crescentum |
| CN114214225B (en) * | 2021-11-28 | 2023-12-15 | 中国人民解放军军事科学院军事医学研究院 | A method for enriching holdfast secreted by Caulobacter crescentus |
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